The serine proteases of the trypsin-like family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Moreover, many serine proteases are involved in diseases such as cancer, wound healing, arthritis, skin diseases, ALS, infection, . . . . All the members of the S1 family have a similar protein fold with a catalytic site consisting of the oxyanion hole and critically important Ser, His and Asp amino acids (the catalytic triad). Typically, the members of this family have a S1 pocket, with at the bottom of this pocket a negatively charged Asp residue.
Kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. KLKs are novel cancer biomarkers. KLKs are of clinical value to identify low- versus high-risk cancer patients, and to predict the course of the cancer disease and response to cancer therapeutics of male and female patients afflicted with reproductive tract malignancies, in addition to cancers of the lung, brain, skin, head and neck, kidney, urinary bladder, and the gastrointestinal tract. Especially in high-risk cancer patient groups, these proteases cannot only be biomarkers for prognosis and therapy response but also act as valuable targets for cancer therapeutics, eventually resulting in reduction of the process of tumor cell dissemination and metastasis.
KLK4 is implicated in cardiovascular diseases, cancer, endocrino-logical diseases, metabolic diseases, gastroenterological diseases, inflammation, hemato-logical diseases, respiratory diseases, neurological diseases, reproduction disorders and urological diseases (WO2005083110). Following Schmitt et al. (2013), Kallikrein-related peptidase 4 (KLK4) is a trypsin-like serine protease displaying arginine/lysine-specific protease activity, with a strong preference for Arg at the P1 position of substrates. In general, KLK4 shares several substrates with other members of the KLK family. The KLK4 gene, also known as prostase or KLK-L1, was initially deemed to be expressed exclusively in the prostate, based on Northern blotting data; however, subsequent studies using RT-PCR demonstrated that KLK4 transcripts are also detected in other tissues, including the testes, mammary glands, adrenals, uterus, thyroid, and salivary glands, although at much lower levels. Notably, KLK4 mRNA overexpression was observed in ovarian carcinoma and shown to constitute an independent indicator of poor prognosis in patients with well or moderately differentiated ovarian tumors. KLK4 gene transcription is also elevated in prostate cancer, compared to normal prostatic epithelium and benign prostatic hyperplasia. As described in WO02077243, KLK4 is implicated in hormone-associated carcinomas, such as breast and prostate cancer. Moreover, KLK4 mRNA expression is an unfavorable prognostic predictor in breast cancer patients. According to the results of a recent study, KLK4 may represent a novel endogenous activator of protease-activated receptor 1 (PAR1), as KLK4 was shown to be aberrantly expressed in colonic tumors and capable of inducing PAR1 signaling in HT-29 colorectal adenocarcinoma cells, thus promoting ERK1/2 activation (Schmitt, Magdolen et al. 2013). Furthermore, KLK4 activates protease activated receptor 2 (PAR2) (Blaber et al. 2010), an activator of inflammatory pathways. As such, KLK4 is a target for the treatment of inflammatory diseases.
The overexpression of KLK4 in the mentioned tumor types clearly demonstrates that KLK4 is a therapeutic target for new drugs.
In 2008 Oikonomopoulou at al. reported a tool using a peptidic activity-based probe coupled antibody capture (Oikonomopoulou, Hansen et al. 2008). The probe was built around a pro-Iys peptidic fragment which was responsible for the recognition of kallikrein 6. Experts in the field will agree that the antibody approach was needed to cope with the selectivity problems related to the probe described by Oikonomopoulou et al. The probe was earlier described by Pan et al. (Pan, Jeffery et al. 2006). This article clearly showed the non-selectivity of the probes. The apparent affinity Ki(app) for tryptase, trypsin, thrombin and plasmin was in the same order of magnitude (0.6 to 6 μM). The authors clearly stated that the incorporation of a proline residue increased the overall reactivity compared to single amino acid Lys probe. However, this was only a moderate activity increase with no change in the selectivity profile. A more recent publication by Brown et al. reports the synthesis and evaluation of phosphonate ABPs targeting matriptase and thrombin. Both are members of the trypsin fold family of S1A proteases (Brown, Ray et al. 2011). The authors stated that for designing broad-specificity phosphonate ABPs or specific S1A protease ABPs a peptide sequence is required. Additionally the leaving group of the phosphonate peptide sequence, peptide length and peptide stability are marked as key elements for enhancing potency. The most potent peptide containing probe has an IC50 value for matriptase of 0.066 mM and a kapp of 490 M−1S−1, which is rather modest. Moreover, the IC50 values of the presented probes are obtained after a long pre-incubation period (4 hours), which emphasizes the slow reaction characteristics.
Most of the small molecule inhibitors that have been employed in biochemical studies of KLKs are often of insufficient potency and/or specificity. Thus, more potent and selective drug like inhibitors are needed (Goettig, Magdolen et al. 2010).
In the above-mentioned context, it is generally recognized that a chemical which could visualize or capture KLK4 catalytic activity on a qualitative and quantitative manner in in vitro and in vivo settings will be highly useful for different non-clinical and clinical applications (Blum, Weimer et al. 2009). The compounds of the present invention allow such a therapeutic and diagnostic (biomarker) application.
The present invention provides the required chemical template (phenyl guanidine, diphenyl phosphonate and specific tail containing a heteroatom) which is a necessity to obtain selective and potent (preferably irreversibly) binding inhibitors or activity based probes directed towards KLK4.
Previously, no diphenyl phosphonate KLK4 inhibitors were reported. A closely relating compound is compound 5a which was originally reported by Sieniczyk et al., 2006 as a uPA inhibitor with limited potency.

We have now surprisingly found that compound 5a is a potent KLK4 inhibitor, however the selectivity profile is not optimal because it inhibits also KLK8, KLK2 and KLK1 (see table 2). Moreover compound 5a shows a reversible binding mechanism against KLK4 instead of the theoretically expected irreversible binding mechanism due to the diphenyl phosphonate group. The described invention under this new patent application is related to further structural improvements to obtain more selective compounds and/or switching the reversible mode of binding towards an irreversible one. These improvements can be pointed to the ideal combination of the phenylguanidinyl moiety with the optimal R3 group. In particular, it was found that the incorporation of a heteroatom, with at least 4 atoms located between the heteroatom and the nitrogen to which R3 is attached, delivered potent irreversible KLK4 inhibitors. In compound 5a such heteroatom is not present at this position, and this compound was found to be a reversible, instead of an irreversible KLK4 inhibitor.
Another series of diphenyl phosphonates described originally by Joossens et al., 2007 belongs to the benzylguanidinyl series which show very potent and irreversible uPA binding (see also WO 2007045496). Nevertheless, we found recently that these compound series are also responsible for potent KLK4, KLK8, KLK2 and matriptase binding (compounds uPA1 and uPA2 of table 2). We understand that this binding profile is very interesting to investigate the potential of those compounds as an anti-cancer drugs, but in general drug developers aim for high selectivity towards the selected target.
The presented compounds in this invention are the first reported diphenyl phosphonate KLK4 inhibitors that show an improved selectivity profile with no potent inhibition against uPA, KLK8, and matriptase. Moreover, we have identified novel compounds which show an irreversible KLK4 binding mode, which seems not evident to obtain within this series, because most compounds are unexpectedly reversible binders. The high selectivity profile of these compounds is very important in their use as activity based probes towards KLK4.